Tuberculosis is an extremely successful pathogen with a penetrance of its host population, mankind, that is estimated by WHO to be close to 1/3 of the population of the planet. In areas of high endemnicity for HIV infection, such as Sub-Saharan Africa, tuberculosis is invariably the first infection to emerge as immunocompetence wanes. Tuberculosis is a serious additional burden to populations already suffering poverty, malnutrition and other infectious diseases. The situation is exacerbated further by the emergence of drug-resistant strains and the absence of new drugs moving into clinical application. This proposal details the development of a high-throughput screen (HTS) platform that will facilitate screening of compounds against M. tuberculosis inside macrophages. This cell-based screen will preserve the conditions experienced by the bacterium inside its host, thereby identifying targets important during the sustenance of infection: 1. Development of real-time reporters of bacterial fitness suitable for a HTS platform. Reporters will either be based on identification of promoters that are activated under situations of duress and used to drive expression of GFP and luciferase as readouts of bacterial fitness, or the use of constitutively-expressed luciferase provided exogenous substrate after exposure to drugs/compounds to identify viable organisms. 2. Optimization of reporter assays for a HTS platform. Once we have developed these reporter strains we will optimize their performance for application in a HTS platform. This includes the specificity of the response, its sensitivity and dynamic range, and its performance in a 96 and 384 well plate format. Once optimized, we will perform the HTS at Dr. Clifton Barry's facility at NIAID. Dr. Barry is a member of the NIH Chemical Genomics Center. 3. Development of secondary assays to evaluate hits, and identify targets and lead compounds. Hits from the primary screen need to be validated and evaluated in a series of secondary screens and relevant compounds need to be analyzed further for identification of the bacterial targets. The goal is to identify some lead compounds that can be developed by our pharmaceutical partners. PUBLIC HEALTH RELEVANCE: Project Narrative / Relevance This proposal is in answer to the Program Announcement PA-07-320, which calls for "Development of assays for high-throughput drug screening". It details approaches for identification of compounds that are bactericidal to Mycobacterium tuberculosis inside macrophages, which mimics its environment in vivo. Tuberculosis continues to be a global threat to mankind, and is named specifically by the NIAID in this announcement. Recent advances have placed us in a strong position to use our scientific advances to design and execute a new HTS platform to identify small molecules capable of killing intracellular M. tuberculosis, which can then be used to develop new drugs or combinations of drugs to more effectively treat this disease.